1. Field of the Invention
The present invention relates generally to the study of promoter hypermethylation. More specifically, the present invention provides a method to overcome incomplete bisulfite modification of DNA recovered from formalin-fixed tissue samples.
2. Description of the Related Art
In higher order eukaryotic organisms, DNA is methylated only at cytosines located 5′ to guanosine in the CpG dinucleotide. CpG rich areas (CpG islands) comprise about 1% of vertebrate genomes and account for about 15% of the total number of CpG dinucleotides. CpG islands are typically between 0.2 to about 1 kb in length and are located upstream of many housekeeping and tissue-specific genes, but may also extend into gene coding regions.
Methylation of cytosine residues within CpG islands plays important role in gene inactivation, cell differentiation, tumorigenesis, X-chromosome inactivation, and genomic imprinting. Extensive methylation of CpG islands has been associated with transcriptional inactivation of selected imprinted genes and genes on the inactive X chromosome of females. CpG islands of constitutively-expressed housekeeping genes are normally unmethylated in the germline and in somatic tissues. Tissue-specific genes are usually unmethylated in the receptive target organs but are methylated in the germline and in non-expressing adult tissues.
Aberrant methylation of normally unmethylated CpG islands has been described as a frequent event in immortalized and transformed cells and has been frequently associated with transcriptional inactivation of tumor suppressor genes in human cancers. Increased methylation of CpG islands associated with tumor suppressor genes may lead to progressive reduction of normal gene expression resulting in the selection of a population of cells having a selective growth advantage (i.e., a malignancy). It is considered that an altered DNA methylation pattern, particularly methylation of cytosine residues, causes genome instability and is mutagenic.
In recent years, emerging evidence indicates that epigenetic alterations associated with promoter hypermethylation is one of the most common molecular events in human neoplasia and this epigenetic change works closely with genetic alterations (coding-region mutation) in human carcinogenesis. Loss of gene function due to promoter hypermethylation has characteristics that mimic the loss of tumor-suppressor gene function due to genetic alterations. Hypermethylation of one allele is frequently accompanied by deletion of the opposite allele, resulting in loss of heterozygosity of the gene. Gene inactivation associated with promoter hypermethylation is fully heritable and loss of gene function due to promoter hypermethylation leads to selective growth advantage in a manner identical to loss of tumor-suppressor gene function.
A number of housekeeping genes are inactivated primarily via promoter hypermethylation in human cancers. A housekeeping gene is a gene of general importance and expresses in a wide variety of organs and tissues. Characteristically, a housekeeping gene contains CpG islands and is devoid of TATA or CAAT boxes in its promoter. The expression of these CpG island-containing housekeeping genes is regulated frequently via promoter CpG island methylation. Genes involved in DNA repair are functionally very important as reflected by their expression in a wide variety of tissues. DNA repair genes, such as hOGG1, hMLH1, MGMT, a gene specific for repair of O6-methylguanine, and NTH1, a gene specific for repair of thymine glycol, all contain CpG islands in their promoter, indicating that these important DNA repair genes are subjected to regulation by promoter methylation.
Methylation-specific PCR (MSP) has recently been developed and used to analyze promoter methylation status of various genes (Herman et al., 1996). One fundamental element of the methylation-specific PCR is to modify DNA using sodium bisulfite before the PCR reaction. In this process, all cytosines not in the CpG sequence (non-CpG cytosines) will be converted to thymine because they are all inevitably free of methylation. On the other hand, cytosines in the CpG sequence (CpG cytosines) will remain as cytosine if the C is methylated but will be converted to thymine if the C is not methylated. This method is considered to be the most sensitive and specific method for promoter methylation determination.
Currently, the methylation-specific PCR method is primarily used with DNA samples extracted from fresh tissues or cells in culture and is only applied sporadically to formalin-fixed, paraffin-embedded tissues. DNA extracted from formalin-fixed tissues is of poor quality and may still contain formalin-induced DNA/DNA or DNA/protein crosslinks that survive the extraction processes. Thus, the prior art is deficient in a method of analyzing promoter methylation in DNA isolated from formalin-fixed, paraffin-embedded tissues. The present invention fulfills this long-standing need and desire in the art.